Role of microtubules in estradiol-17β-d-glucuronide-induced alteration of canalicular Mrp2 localization and activity

Abstract

(E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 mol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 mol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicinepretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process. bile secretion; endocytic compartment; colchicine; bilirubin THE MULTIDRUG RESISTANCE-ASSOCIATED protein 2 (Mrp2; Abcc2) mediates the ATP-dependent transport of a wide range of amphiphilic anionic conjugates into bile (5,35

Radixin Is Required to Maintain Apical Canalicular Membrane Structure and Function in Rat Hepatocytes

“…Mrp2, as well as Bsep and Mdr1, have all been identified in intracellular vesicular structures in addition to the canalicular membrane in rat liver 20,21 . Furthermore, Mrp2 and Bsep are regulated rapidly by retrieval from and/or insertion into the canalicular membrane via intracellular vesicles 21,22,23,24,25 . In support of our finding, a recent study in WIF-B9 cells colocalized apical ABC transporters and Rab11 in polarized cells and restricted these transporters to Rab11-endosomes in non-polarized cells where no canalicular pole was formed 27 .…”

Molecular Mechanism of Altered Ezetimibe Disposition in Nonalcoholic Steatohepatitis

“…Blocking of biliary transport by way of altered localization of efflux drug transporters could, in conjunction with increased expression of Abcc3 on the sinusoidal membrane, drive the shift from biliary to plasma efflux. Likewise, Mottino et al (2002Mottino et al ( , 2005 have shown that altered localization of Abcc2 during estradiol-17␤-glucuronide-induced cholestasis results in reduction of the biliary concentration of Abcc2 substrates. Zhang et al (2005) observed a similar phenomenon in sandwich-cultured rat hepatocytes.…”

EGFR participates downstream of ERα in estradiol-17β-d-glucuronide-induced impairment of Abcc2 function in isolated rat hepatocyte couplets

“…Our group has recently demonstrated that GPR30, a GPCR, and ERα accounts in part for the acute cholestasis caused by E17G Barosso et al 2012), event that correlates well with its ability to induce endocytic internalization of the canalicular transporters such as Abcc2 (Mottino et al 2003(Mottino et al , 2005. The actions of GPR30 and ERα in E17G-induced cholestasis constitute two independent pathways.…”

“…Cells were examined with a Nikon C1 Plus confocal laser scanning microscope, attached to a Nikon TE-2000 inverted microscope. Densitometric analysis of images was made along a line perpendicular to the canalicular vacuole using the Image J 1.44p software (Improvision, Coventry, NIH), as previously described for liver tissue slices (Mottino et al 2005). Each measurement was normalized to the sum of all intensities of the respective measurement.…”

“…Canalicular width was estimated as the width of the profile of actin associated fluorescence at half its maximal fluorescence value. Only canaliculi with a width between 1 and 2.5 µM were analyzed (Mottino et al 2005).…”